The goal of this project is to develop and evaluate methods for manufacturing dendritic cells (DCs) for clinical immunotherapy trials. In FY 1999, we developed and optimized a full-scale GMP method for 5-day flask culture of autologous DCs in RPMI, autologous plasma or allogeneic serum, IL4 and GMCSF, starting with peripheral blood monocytes collected by apheresis and purified by elutriation. The immature DCs generated are then available for further manipulations (e.g., peptide pulsing) prior to clinical administration. This manufacturing method was incorporated into several clinical trials, and a manuscript describing this method was published in early 2001. In FY2000, because of our interest in developing closed systems and eliminating reagents that are difficult to standardize, we evaluated a 7-day culture system in a protein-defined, serum-free medium (XVIVO15) starting with monocytes from elutriation vs. negative immunomagnetic selection using the Isolex 300I, in bags vs. flasks. We demonstrated that the 2 different isolation methods for monocytes product equivalent immature DC populations, and that bags were equivalent to flasks. Furthermore, historical comparison showed that serum-free medium was equivalent and perhaps even superior to serum-containing medium for generation of immature DCs. A manuscript describing this work was published in January 2002. In FY2001, we focused on evaluating culture conditions for generating mature DCs using CD40 ligand after culture in IL4 and GMCSF. A process was successfully developed and incorporated into several cancer immunotherapy trials in early 2001. During FY2002, we completed studies demostrating stability after overnight hold at 4C of the raw material (mononuclear cell concentrates) in terms of ability to generate immature and mature DCs. We also completed stability testing of mature peptide-pulsed DCs and established a 2 hour room temperature hold period to accomodate transport or delays in administration of the product. In addition, we established a program to document lot-to-lot potency of the CD40ligand reagent used to mature DCs in culture. Ongoing studies are focused on characterizing DCs by flow cytometric phenotyping. In FY2003, a pilot study evaluating the feasibility of DC cryopreservation was completed. Mature DCs generated by the method previously described were successfully cryopreserved in 5% DMSO and 6%Pentastarch, with 5% human serum albumin in Plasmalyte A. Higher protein concentrations did not significantly improve DC recovery after the freeze/thaw process. Additional cryopreservation studies are planned.